Mechanisms of Hedgehog Target Gene Selection in Development and Cancer

描述(申请人提供)：刺猬(HH)信号是控制分化和生长的基础。在小脑发育过程中，浦肯野神经元发出Sonic hedgehog(Shh)，这是邻近颗粒神经元前体(GNPs)的有效有丝分裂原。GNP通过改变Gli转录因子的加工、定位和修饰来响应Shh，Gli转录因子激活或抑制靶基因。人类或小鼠Patched1中编码Shh受体的突变促进了GNP细胞向髓母细胞瘤(MBS)的转化，MBS是最常见的儿童恶性脑瘤。我们使用染色质免疫沉淀(CHIP)和高通量测序来确定GNPs和MB细胞染色质中Gli1结合的位置。这让我们找到了Gli反应转录增强子。结合芯片数据和基因表达数据，我们确定了Shh直接调控的可能靶基因。当小脑前体细胞成为癌细胞时，Gli1的靶向和靶基因的表达发生了戏剧性的变化。我们将研究靶基因选择的机制，HH靶基因与细胞周期的关系，以及靶基因在正常发育和肿瘤发生中的作用。具体目的1：研究Gli转录因子在小脑发育和肿瘤发生中如何协同调控基因表达。我们的芯片数据导致了许多新的靶基因，以及ptch1、Gli1和N-myc等成熟的靶基因。132个基因在正常和肿瘤细胞类型中是一致的靶点。值得注意的是，尽管GNPs与MB细胞关系密切，但许多假定的靶基因是针对一种或另一种细胞类型的。我们将确定在这两种细胞类型中选择性识别增强子元件的机制。具体目标2：确定染色质修饰物如何影响Gli调节的基因表达。我们已经确定了组蛋白修饰与GNPs与MBS中特定目标基因的调控变化相关。我们将研究HH靶基因规范中这些变化的机制重要性。具体目的3：研究Gli蛋白与其他转录因子的相互作用。对Gli1结合的DNA区域的计算分析揭示了两种类型的转录因子的证据，即E盒结合蛋白和NFI蛋白，它们可能与Gli1蛋白平行工作，也可能协同工作。我们将研究它们在靶基因调控中的作用。具体目标4：确定Gli靶标在GNP发展和肿瘤发生中的作用。我们将在GNPs和肿瘤的背景下，研究调节HH信号和细胞周期之间联系的选定的目标基因。计划中的研究对于了解Hedgehog通路在许多组织和器官中的发育和致癌作用具有直接重要意义。发现由HH信号直接调节的基因将导致当信号转导错误导致出生缺陷或癌症时进行干预的新方法。 公共卫生相关性：Hedgehog(HH)途径对许多器官和组织的构型、增殖和分化至关重要。在小脑，HH信号通过激活和抑制靶基因来控制颗粒神经元的产生。放松对HH靶基因的调控通常会导致细胞分裂不受限制，最终导致髓母细胞瘤，这是最常见的儿童脑部恶性肿瘤。利用基因组学方法，我们发现在小脑正常发育和肿瘤形成期间，HH作用的转录景观是截然不同的。我们将确定选择HH靶基因的分子机制，研究新发现的辅因子，并确定靶基因如何控制正常和肿瘤的生长。

DESCRIPTION (provided by applicant): Hedgehog (Hh) signaling is fundamental to the control of differentiation and growth. During development of the cerebellum, Purkinje neurons emit Sonic hedgehog (Shh), a potent mitogen for adjacent granule neuron precursors (GNPs). GNPs respond to Shh by altering the processing, location, and modification of Gli transcription factors that activate or repress target genes. Mutations in human or mouse patched1, which encodes the Shh receptor, promote transformation of GNP cells into medulloblastomas (MBs), the most common childhood malignant brain tumor. We used chromatin immunoprecipitation (ChIP) and high-throughput sequencing to identify locations of Gli1 binding in the chromatin of murine GNPs and MB cells. This led us to Gli-responsive transcriptional enhancers. Combining ChIP data with gene expression data we identified putative target genes that are directly regulated by Shh. Dramatic changes in targeting of Gli1, and target gene expression, occur when cerebellum precursor cells become cancer cells. We will investigate the mechanisms of target gene selection, the connections between Hh target genes and the cell cycle, and the roles of target genes in normal development and tumorigenesis. Specific Aim 1: Investigate how Gli transcription factors coordinate to regulate gene expression in cerebellum development and tumorigenesis. Our ChIP data led to many novel target genes, and well-established targets like Ptch1, Gli1, and N-myc. 132 genes are consistent targets in normal and tumor cell types. Remarkably, despite the close relation between GNPs and MB cells, many putative target genes are specific to one cell type or the other. We will determine the mechanism of selective recognition of enhancer elements in the two cell types. Specific Aim 2: Determine how chromatin modifiers influence Gli-regulated gene expression. We have identified histone modifications that correlate with the regulatory changes for specific target genes in GNPs vs. MBs. We will investigate the mechanistic importance of these changes in Hh target gene specification. Specific Aim 3: Investigate interactions of Gli proteins with other transcription factors. Computational analyses of the DNA regions bound by Gli1 revealed evidence for two types of transcription factors, E box-binding proteins and NFI proteins, that may work in parallel, or collaborate, with Gli1 protein. We will investigate their roles in target gene control. Specific Aim 4: Define contributions of Gli targets to GNP development and tumorigenesis. We will investigate selected target genes that mediate the connection between Hh signaling and the cell cycle, in the context of GNPs and tumors. The planned studies have direct importance for understanding developmental and tumorigenic roles of the Hedgehog pathway in many tissues and organs. Discovering genes that are directly regulated by Hh signaling will lead to new ways to intervene when errors in signal transduction lead to birth defects or cancer. PUBLIC HEALTH RELEVANCE: The Hedgehog (Hh) pathway is critical for patterning, proliferation, and differentiation in many organs and tissues. In the cerebellum, Hh signaling controls production of granule neurons by activating and repressing target genes. Deregulation of Hh target genes often results in unrestrained cell division and eventually medulloblastoma, the most common pediatric brain malignancy. Using genomic methods, we have found that the transcriptional landscapes of Hh action are strikingly distinct during normal development and tumor formation in the cerebellum. We will determine the molecular mechanisms by which Hh target genes are chosen, investigate newly discovered cofactors, and determine how target genes control normal and tumor growth.